JP2008505071A - Methods for genetically activating cells and uses of the cells - Google Patents

Abstract

Normally, IL-2 dependent mammalian cells can be successfully transfected to express IL-2 in an amount sufficient to maintain proliferation without the addition of external IL-2. One is a cell line that expresses IL-2 only in the endoplasmic reticulum but does not secrete, and the other is a cell line capable of secreting IL-2, which were developed and tested. Preliminary experiments using primary cells from human donors confirmed the feasibility of the present invention. The present invention provides genetically modified cells, methods for their production, as well as methods for treating cancer and promoting immunity.
[Selection] Figure 1

Description

  The present invention relates generally to genetic engineering, the production of genetically modified cells, and immune promotion, specifically their use in the treatment of cancer.

  Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that are clearly distinguishable from T and B lymphocytes [1]. They play an important role in innate immune responses against many pathogenic microorganisms [2] [3]. In addition, they mediate strong anti-tumor responses, as demonstrated in several in vitro [4] and in vivo [5] experimental models. In vivo, they can control proliferation and metastatic spread [6]. NK cells can also contribute to resistance to human malignant tumors, which has been clearly demonstrated in the context of stem cell transplantation to treat hematological malignancies. With these observations, some are aimed at enhancing the activity of human NK cells in cancer patients in vivo, for example by using specific cytokines or other stimuli that directly enhance the activity of NK cells in vivo. Such research has been promoted [7].

  Antitumor effects mediated by NK cells are enhanced by cytokines such as interleukin 2 (IL-2), IL-12, IL-15, IL-18, and IL-21 [8-11] [12] be able to. A number of attempts have been made to systemically administer IL-2 to cancer patients. Such a strategy has resulted in a mixed clinical outcome that depends on the protocol, the type and stage of the cancer, and other factors. However, systemic administration of IL-2 is often accompanied by undesirable side effects [13-15], such as toxicity affecting the cardiovascular, gastrointestinal, respiratory and nervous systems. The latter includes thought disorders, emotional changes, loss of appetite, and flu-like symptoms. In situations where IL-2 is primarily given to enhance NK activity, it would be ideal to administer in a form that stimulates NK cells but does not cause any undesirable side effects. As such, research on alternative approaches for IL-2 delivery has been underway.

  NK-92 is an NK cell line grown in vitro with the phenotypic and functional characteristics of primary human NK cells [16]. This cell line is strictly IL-2 dependent on proliferation and survival. Therefore, they are utilized as a particularly useful model for studying IL-2 action in NK stimulation. Several attempts have been made to transduce functional IL-2 genes into NK-92 cells or introduce them by other means. These studies have shown that IL-2 expressed by these means fulfills the goal of stimulating NK-92 proliferation and survival.

  The main objective underlying the present invention is to find an alternative to systemic IL-2 administration, in addition to culturing IL-2-dependent cells such as T cells and NK cells ex vivo. To eliminate the need for IL-2 addition. One of our specific objectives is to generate self-activated NK cells. Other objects and goals and solutions provided by the present invention, as well as their associated advantages, will be apparent to those skilled in the art upon studying the detailed description, non-limiting examples, and claims. It is expected to become.

  We have surprisingly found that IL-2 dependent mammalian cells are IL-2 (specifically, in an amount sufficient to maintain proliferation without the external addition of IL-2. Was found to be successfully transduced so that IL-2 is expressed). In our study, we cloned three different forms of IL-2 and expressed them in cultured mammalian cells. The expression of this construct was verified by immunostaining of transduced Cos-7 cells. The biological activity of the modified protein was evaluated in NK-92 cells. Surprisingly, the endoplasmic reticulum (ER) retained sufficient IL-2 type to promote NK-92 cell proliferation and survival. Moreover, the cells expressed in this way had cytotoxic activity. This study demonstrates that it is possible to express IL-2 in NK-92 cells in such a way as to prevent cytokine secretion and thus any possible unwanted side effects. The contents of the discovery of the present invention were considered.

  The present invention relates to cells that express genetically modified IL-2, methods for their production, as well as methods of treatment, as defined in the appended claims included herein by reference. For example, providing immune stimulation or immunotherapy.

  The present invention is illustrated by examples showing that the recombinant NK-92 cell line, NK92IL2ER, expresses IL-2 at a level sufficient for survival of NK cells in a limited region of the endoplasmic reticulum. NK92IL2WT, another genetically engineered NK-92 cell line, can secrete IL-2 in an amount comparable to that of unmodified activated T cells. Preliminary experiments conducted during the priority year indicate that the results can be transduced into human primary cells obtained from healthy donors.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is described in more detail in the following detailed description, examples and the accompanying drawings:
FIG. 1 shows a schematic diagram of a plasmid expressing a modified form of IL-2. L: leader; IL-2: interleukin 2; ERRS: endoplasmic reticulum retention signal.
FIG. 2 is a bar graph showing the growth of IL-2 transduced NK-92 cells and parental NK-92 cells in short-term (6 days) culture. The NK-92 cells, the initial concentration of 25,000 cells ^{/ ml, 90% CellGro (R} ), 10% FBS, and were cultured in 500 IU / ml of IL-2. On day 0, the cells were washed thoroughly with PBS to remove any small amounts of IL-2 from previous culture steps. Unmodified NK-92 cells died in the absence of IL-2, but genetically modified cells grew as well as cells in the presence of added IL-2.
FIG. 3 is a bar graph showing the results of co-culturing NK-92GFP and NK-92 modified cells expressing IL-2 at a ratio of 1: 1. NK-92 cells expressing 25000 IL-2 were mixed with NK-92 cells expressing the same number of GFP. Both cell groups were thoroughly washed twice with PBS to eliminate any external small amounts of IL-2 from previous cultures. After 48 hours, the ratio of GFP positive cells to negative cells was quantified by FACS analysis. In culture with NK-92IL2ER, NK-92GFP cells died, demonstrating the absence of IL-2 secretion from these cells.
FIG. 4 is a bar graph illustrating IL-2 production of genetically modified NK-92 in vitro. Cells were cultured for 6 days at a starting concentration of 25000 cells / ml. The supernatant was collected 48 hours after the medium was changed. The level of IL-2 in the supernatant was measured using an enzyme linked immunosorbent assay (ELISA) as described in the Materials and Methods section. The results show that NK92IL2WT IL-2 production and secretion is comparable to that of T cells, whereas IL-2 levels in the supernatant of NK92IL2ER cells are comparable to medium alone (control). Is shown.
FIG. 5 shows the cytotoxicity of parental and genetically modified cells against K-562 cells in a 4 hour ⁵¹ Cr release assay. Genetically modified cells retain the same cytotoxic performance as unmodified cells.

  Before describing the present invention, it will be understood that the scope of the present invention is limited only by the appended claims and their equivalents, and thus terms used herein merely describe specific embodiments. It is used only for the purpose and not for the purpose of limitation.

  The terms “treatment”, “treatment”, “therapeutic use”, “pharmaceutical” and “medical use” encompass both human and animal or veterinary uses.

  The term “functional equivalent” defines a protein or nucleotide sequence that has a different amino acid or base sequence compared to the sequences disclosed herein, but that exhibits the same function in vitro and in vivo. An example of a functional equivalent is a modified or synthetic gene that encodes the expression of a protein that is identical or highly homologous to the protein encoded by the wild type gene.

  “Transfection” means gene transfer by physical or chemical means. “Transduction” means gene transfer by a viral vector. “Stable genetic modification” means that the genetic modification is inherited genetically, which generally (but not necessarily) has been transfected into any one of the cell's chromosomes. This is achieved by incorporating DNA. "Transient genetic modification" means a loss of genetic material over a time course of weeks or months.

According to an embodiment, the present invention provides a method for producing a mammalian cell expressing IL-2, the method comprising the following steps:
Selecting or constructing an IL-2 gene;
Producing a retroviral vector having the IL-2 gene;
Recover cells from the donor,
Genetically modifying the cell; and
Optionally selecting transfected cells,
Consists of.

  The IL-2 gene is preferably a gene encoding the protein represented by SEQ ID NO: 11 or a functional equivalent thereof. The IL-2 gene is more preferably a gene encoding the protein represented by SEQ ID NO: 13, or a functional equivalent thereof. Most preferably, said gene is selected from SEQ ID NO: 8, SEQ ID NO: 10, or functional equivalents thereof.

  According to one embodiment, the mammalian cell is selected from natural killer (NK) cells and T cells, preferably natural killer (NK) cells.

  According to another embodiment, the modified IL-2 gene is modified such that IL-2 is expressed towards the endoplasmic reticulum of the cell.

  The invention further provides a transgenic mammalian cell capable of producing IL-2, produced by the methods outlined above and further specified in more detail in the Examples.

  Although the present invention specifically is IL-2 dependent on proliferation in the untransfected state and cannot produce them in significant quantities, these transgenic cells are exogenous. It relates to mammalian cells that produce IL-2 in an amount sufficient to maintain proliferation without the need for IL-2.

  According to a preferred embodiment, IL-2 expression is limited to the endoplasmic reticulum of said cell. For example, simply preventing IL-2 secretion by removing the secretion signal (Construct 2, FIG. 1) is not sufficient to provide autocrine growth stimulation to genetically modified cells, It is important to note that.

  The present invention also provides therapeutic methods such as elective, curative and prophylactic treatment. According to one embodiment, the present invention relates to a method for the treatment of cancer, which method comprises administering to said patient a therapeutically effective and physiologically acceptable amount of a transgenic cell as defined above. . The cancer may be in any form of cancer, such as, but not limited to, colon cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, stomach cancer, lung cancer, brain tumor, and skin cancer (black) Tumors), including leukemias and lymphomas. Currently, the effect of IL-2 administration has been demonstrated primarily in metastatic kidney cancer and metastatic melanoma.

  The present invention also provides a method of promoting immunity using genetically modified cells that express substantially physiological levels of IL-2, in which the mammal expresses IL-2. The cell comprises the following steps: selecting or constructing the IL-2 gene; producing a retroviral vector having the IL-2 gene; collecting the cell from a donor or patient; genetically modifying the cell; Optionally produced by a method comprising selecting a genetically modified cell and administering the modified cell to a patient in need thereof.

  As described above, the IL-2 gene is preferably a gene encoding the protein represented by SEQ ID NO: 11 or a functional equivalent thereof. The IL-2 gene is more preferably a gene encoding the protein represented by SEQ ID NO: 13, or a functional equivalent thereof. Most preferably, said gene is selected from SEQ ID NO: 8, SEQ ID NO: 10, or functional equivalents thereof.

  The present invention also provides a method of stimulating a patient's immune system comprising administering to said patient a therapeutically effective and physiologically acceptable amount of a transgenic cell as defined above. Including. It is envisaged that the immune promotion or immunotherapy is one of the steps in cancer treatment or infection treatment or prevention. As defined above, the cancer may be in any form of cancer, such as, but not limited to, colon cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, stomach cancer, lung cancer, Examples include brain tumors and skin cancer (melanoma), including leukemia and lymphoma.

  In the above method, the cells are preferably harvested from a patient, genetically modified and returned to the same patient. However, it is envisioned that the cells are collected from a donor, genetically modified, and administered to the patient.

  The use of the transgenic cells may be one of additional or supplemental treatments that are performed before, after, or substantially simultaneously with other treatments. In the treatment of cancer, the transgenic cells can be administered before, after, or substantially simultaneously with cytotoxic drugs, radiation therapy, surgical intervention or combinations thereof. However, it is also envisaged that the use of said transgenic cells is one of the primary therapies in the treatment of cancer.

  The invention also encompasses the use of said cells for the manufacture of a pharmaceutical composition or medicament for use in the treatment of cancer or the treatment or prevention of infection. The type of cancer may be any type of cancer, but is preferably one of the types of cancer defined above.

According to the study by the present inventors, by adding an ER retention signal to the cytokine coding sequence, IL-2, which is a cytokine that acts on the whole body, is strictly given an autocrine signal transduction mode. It is demonstrated that it is possible. The NK-92 natural killer cell line genetically modified by retroviruses continues to grow in the absence of exogenously added IL-2. Previous studies [26, 27] have reported the modification of the NK-92 gene by stable transfection. TR-IL-2-NK- 92 cells were produced in 5.5ng / 10 ^6/24 hours, NK-92MI is produced at 1260pg / ml / 48 h / 10 ^6, NK-92CI is 15 pg / Production in ml / 48 hours / 10 ⁶ . Some of these cell lines produce significantly higher levels of IL-2 compared to primary cells, which can be explained by the incorporation of multicopy plasmids during transfection, and in some cases May be considered harmful. NK-92IL2WT of the present invention is produced at 18.3 pg / ml / 48 hours / 10 ⁶ , which is equivalent to activated primary human T cells (40 pg / ml / 48 hours / 10 ⁶ ).

  Secretion of constructs targeting ER was comparable to background, but cells grew well. This surprising finding can be explained by ER-retained IL-2 binding to its receptor in the ER up to the cell membrane. It is also possible that signal transduction occurs from the receptor-ligand complex directly from the ER. Similar modes of signal transduction have been described for GM-CSF [29] and IL-3 [30].

  Systemic administration of IL-2 to a patient to retain transfected immune effector cells is associated with strong side effects. Delivering NK cells that can promote their own proliferation as IL2ER-cells causes cytokines (eg TNF-a and TNF-a) that are naturally produced by activated NK cells without causing the side effects of systemic injection. Provides stimulation to surrounding immune cells via IFN-g) and further local secretion of IL-2 (IL2WT cells), which is advantageous for direct anti-tumor effects in cancer immunotherapy Similarly, other applications may be advantageous when immune enhancement is desired.

  Transgenic IL-2 producing cells have a surprising advantage in that they are likely to become resistant to the defense mechanisms of tumor cells (generally silenced NK cells). IL-2 production obtained by gene transfer confers self-stimulating properties to cells.

  In one embodiment, if transgenic IL-2 producing NK cells also secrete IL-2, they stimulate surrounding untransfected cells via IL-2 secreted at physiological levels. Providing localized stimulation to the immune system. This has not been achieved in any other way.

  That is, the present inventors are able to have a direct anti-tumor effect without systemic assistance in the form of IL-2 injection, and in addition to the local production of cytokines naturally produced by NK cells. Provided are genetically modified cells for immunotherapy of cancer that are capable of secretion or localized immune stimulation via additional IL-2. The inventors have shown the ability of ER-retained IL-2 to provide autocrine growth stimuli to genetically modified cells without secreting cytokines into the extracellular compartment.

  Another advantage of the present invention is that it avoids time consuming and expensive ex vivo culture steps. While saving time alone is significant, it is also an important improvement in that the handling of cells administered to patients ex vivo is strictly regulated.

  Furthermore, administration of IL-2 to promote cell proliferation is no longer necessary, whether ex vivo or in vivo. For in vivo applications, the side effects of systemic administration of IL-2 are avoided.

  Other problems of the prior art have been overcome by the methods and modified cells of the present invention, as well as the advantages associated therewith, more detailed description of the invention, examples, figures and accompanying sequence listing. It will be apparent to those skilled in the art upon studying in detail.

〔Example〕
Materials and methods
Cell line phoenix GP was used for cell line retrovirus production (Department of Molecular Pharmacology, Stanford University School of Medicine, Dr. Garry P. Permission from Stanford, CA. ). For immunostaining, cell line Cos-7 derived from African green monkey (DSMZ, Braunschweig, Germany) was used. Both cell lines were treated with Dulbecco's Modified Eagle Medium (DMEM containing Glutamax, sodium pyruvate, 4500 mg / l glucose, and pyridoxine and supplemented with 10% fetal calf serum (FBS-Invitrogen). -Invitrogen, Paisley, Scotland).

The NK-92 cell line was purchased from LGC Promochem / ATCC (LGC Promochem / ATCC, Volos, Sweden). The NK-92 cells, the 10% FBS and 500IU / ml IL-2 (Proleukin (Proleukin ^(R)), Chiron (Chiron), CA, USA) added stem cell medium (Cellgro (CellGro ^(R) )) Maintained in. Cellgro ^(R) SCGM hematopoietic stem and progenitor cells (cell transgenic scan (CellGenix), Freiburg, Germany) for culturing, GMP (standards for production and quality control of pharmaceuticals) is a serum-free medium quality. Proleukin ^(R) is an interleukin-2 GMP quality, which, aliquoted and stored at -20 ° C. at a stock concentration of 10 ⁶ IU / ml.

  As a target for natural killer cells, the human myeloid leukemia cell line K-562 (LGC Promochem / ATCC, Volos, Sweden) was used. K-562 cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS.

All cell lines were incubated at 37 ° C., 5% CO ₂ and 95% humidity and subcultured every 2-3 days. All media was stored at + 4 ° C and FBS was heat inactivated at 56 ° C for 1 hour and stored at -20 ° C. Aliquots of cells from early passages are frozen in 10% dimethyl sulfoxide (DMSO-Sigma-Aldrich, St. Louis, MO, USA) / 90% FBS for later reconstitution. Stored at -150 ° C. Phosphate buffered saline (PBS) free of calcium, magnesium and sodium bicarbonate was purchased from Invitrogen and stored at 4 ° C.

All cell populations were observed at regular intervals using an inverted microscope (Olympus CK40) equipped with a UV module (Olympus U-RFLT50), and cell viability was determined using trypan blue exclusion. In addition, mycoplasma contamination was monitored regularly. A FACScalibur was used with Cell Quest ^™ 3.3 analysis software (Becton Dickinson, Calif., USA) for data collection and analysis. In each sample, at least 10,000 cells were obtained in the analysis area of viable cells defined by side scatter and forward scatter.

The pORF-hIL2 plasmid containing the plasmid IL-2 cDNA template was purchased from In vivoGen (InvivoGen, San Diego, CA, USA). The required IL-2 primers were designed using Oligo 6.6 software (Molecular Biology Insights Inc, Colorado, USA) and they were DNA Technology ApS (DNA (Technology ApS, Aarhus, Denmark). The IL-2 mutant was cloned by PCR using the following primers:
SEQ ID NO: 1: wild type IL-2 [TTA CAA TTG ATC ACC GGC GAA GGA GG] (forward), and
SEQ ID NO: 2: [TTA ATC GAT GTA TCT TAT CAT GTC G] (reverse);
SEQ ID NO: 3: IL-2 [ACC GCC ATG GCA CCT ACT TCA AGT TCT ACA AA] (forward) in the cytoplasm (no leader), and
SEQ ID NO: 4: [TTA ATC GAT GTA TCT TAT CAT GTC G] (reverse); and
SEQ ID NO: 5: endoplasmic reticulum IL-2 [TTA CAA TTG ATC ACC GGC GAA GGA GG] (forward), and SEQ ID NO: 6: [TCA CAG TTC GTC CTT CTC GCT GCC AGT CAG TGT TGA GAT GAT GCT TT] (reverse, Including endoplasmic reticulum retention signal).

The PCR product was cloned into the pCR ^{(R) 4} Blunt TOPO ^(R) vector (Invitrogen). TOPO ^(R) is the cloning and transformation steps were performed according to the manufacturer's instructions. Clones were analyzed using restriction analysis and cycle sequences and subcloned into pSF91-MCSg using EcoRI.

PSF91-MCSg was derived from the murine leukemia virus-based retroviral vector pSF91-GFP-gPRE, kindly donated by Professor Christopher Baum (Hanover Medical School, Hannover, Germany). To facilitate further construction, NotI-HindIII fragments (including GFP and gPRE) are combined at a synthetic oligonucleotide cloning site containing restriction sites for EcoRI, NotI, BamHI, HindIII, NruI, SalI, and MfeI. Replaced (SEQ ID NO: 7). Subsequently, the gPRE element as an EcoRI fragment was reinserted into the MfeI site to create pSF91-MCSg. For eGFP vector construction, the eGFP gene from pEGFP-N3 (Clontech, Palo Alto, Calif.) was released using HindIII-NotI (contained in between) and HindIII- of pSF91-MCSg The plasmid obtained by inserting between SalI (contained in the meantime) was named pSF91-GN3g. All constructs were confirmed by restriction mapping and partially sequenced. Finally, three constructs were produced;
A first sequence (SEQ ID NO: 8) that expresses (secretes) wild-type IL-2;
For comparison, a second sequence (SEQ ID NO: 9) targeted to the cytoplasm was constructed.
Finally, a third sequence (SEQ ID NO: 10) targeted to the endoplasmic reticulum was constructed.
See FIG. 1 for a schematic diagram of these plasmids.

The corresponding amino acid sequence is also shown in the attached sequence listing:
SEQ ID NO: 11: IL-2 wild type SEQ ID NO: 12: IL-2 without leader
SEQ ID NO: 13: IL-2ER.

QIAprep 8 Turbo miniprep (QIAprep 8 Turbo Miniprep), QIAprep 8 miniprep (QIAprep 8 miniprep), and, Qiagen ^(R) plasmid maxi ^{(Qiagen (R) Plasmid Maxi)} ( Qiagen (Qiagen Inc.), California, USA), and in some cases, the plasmid DNA was purified using the Genelute HP Plasmid Midiprep Kit (Sigma-Aldrich). All the above kits were used according to the manufacturer's instructions.

Transfection and transduction Phoenix GP cells and Cos-7 cells are encoded with 3 μg and 2 μg of vector construct plasmid, respectively, and pMD-G (1 μg) (vesicular stomatitis virus envelope glycoprotein per 35 mm cell culture well, Transiently transfected at the University of Geneva (University of Geneva, Geneva, Switzerland), kindly provided by Dr. D. Trono of Department of Genetics and Microbiology (Dept. of Genetics and Microbiology). After transfection of this Phoenix GP, virus supernatant was collected for transduction experiments, and Cos-7 cells transfected for immunostaining were used. For transfection, Fugene 6 reagent (Roche Boehringer Mannheim, Germany) was used according to the manufacturer's instructions. Briefly, the DNA plasmid vector and the Fugen reagent were mixed in cell medium (100 μl volume) at a 1/2 mass / volume ratio and added to the cells after 15 minutes. As a positive control, a plasmid (pSF91-GN3g) containing GFP in the main chain of the retrovirus was used. Supernatants were collected 24 and 48 hours after transfection and filtered through a 0.45 μm Millex-GP syringe top filter (Millipore Corporation, Bedford, Mass.). And immediately used for transduction. The transfection efficiency in the positive control was always higher than 50%.

Using the supernatant containing the above vector, transduced into NK-92 cells, the cells, IL-2 supernatant 300 [mu] l, and, 4 [mu] g / ml bromide hexadiene cytometry Jin (polybrene (Polybrene ^(R)), Sigma Centrifugation at 1000 × g for 1 hour in the presence of -Aldrich).

The biological activity of the proliferated expressed interleukin-2 was measured by cell proliferation analysis using the IL-2-dependent cell line NK-92. Cell proliferation was quantified by counting cells in a Bucher chamber using viability measurement with trypan blue staining and cell proliferation reagent WST-1 (Roche Boehringer Mannheim) according to manufacturer's recommendations.

Quantitative analysis of secreted interleukin-2 in the supernatant of NK-92 cells transduced with IL-2 For quantitative determination of human interleukin-2, OptEIA ^™ human IL-2 ELISA kit II (BD Bio Sciences Pharmingen (San Diego, CA, USA) was used according to the manufacturer's instructions.

Cos-7 cells modified with immunostained IL-2 were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature (RT). After fixing, the cells were rinsed with PBS and incubated with 1% PBS solution of NP40 (Vysis Inc, Illinois, USA) for 10 minutes at room temperature. The cells were then washed 3 times with PBS, 0.1% Tween 20 (Sigma-Aldrich), 0.1% BSA-c (Aurion, The Netherlands), and 5% in PBS. Blocked with blocking buffer containing% goat serum (DAKO A / S, Glostrup, Denmark) for 30 minutes. Cells were washed with PBS / 0.1% Tween 20 three times for 4 minutes and diluted with blocking buffer to 5 μg / ml primary purified rat anti-human IL-2 antibody (BD Biosciences Farmingen) 45 Incubated for minutes. Cells were then washed again 4 times for 5 minutes in PBS / 0.1% Tween 20 and incubated for 1 hour with 5 μg / ml secondary Oregon Green 488 nm goat anti-rat IgG antibody in blocking buffer. Finally, the cells were rinsed with PBS / Tween 20 and diluted 1: 4000 with PBS to Hoechst stain (Molecular Probes BV, Leiden, The Netherlands) for 5 minutes at room temperature. Counterstained and subsequently washed once with PBS. Fluorescence using a Leica DMRXA microscope (Leica Microsystems, GmbH, Wetzlar, Germany) equipped with a CCD camera (Model S / N370KL0565, Cook Corporation, New York, USA) Cells were visualized by microscopy. Filters set to DAPI / Hoechst, FITC, Cy3 and Cy5 were obtained from Chroma (Chroma Technology, Brattleboro, VT, USA). Slidebook 2.1.5 software (Intelligent Imaging Innovations Inc, Denver, Colorado, USA), and Adobe Photoshop 5.0 (Adobe Systems ( Images were obtained using Adobe systems (Seattle, WA, USA).

To confirm that the IL-2 expression field is actually in the endoplasmic reticulum, a second immunostaining was performed. After transduction, NK-92 cells were stained with ER-Tracker ^™ Blue-White DPX (Molecular Probes, Eugene, USA) according to the manufacturer's instructions. The ER-tracker specifically stains the endoplasmic reticulum. After washing with PBS, cells were fixed with 2% paraformaldehyde, permeabilized with NP40, and stained with IL-2 antibody as described above.

NK-92 cells modified to express co-cultured green fluorescent protein (GFP) between NK-92 cells expressing different forms of IL-2 and NK-92 cells modified with GFP Mixed with NK-92 cells modified in 2 at a 1: 1 ratio. Co-culture experiments were performed in 6-well plates as follows: 25,000 NK-92 cells expressing IL-2 were mixed with NK-92 cells expressing the same number of GFP. Both cell groups were thoroughly washed twice with PBS to eliminate any small external amounts of IL-2 from previous cultures. After 48 hours, the ratio of GFP positive cells to negative cells was quantified by FACS analysis.

Cytotoxicity Analysis Cytotoxic function was measured in triplicate with a standard 4 hour ⁵¹ Cr release assay. Briefly, 1 × 10 ⁶ K562 cells were labeled with ⁵¹ Cr (100 μl) and incubated at 37 ° C. for 1 hour. Effector cells were counted using trypan blue exclusion dye and mixed with target cells to obtain effectors: target ratios of 10: 1, 3: 1, 1: 1, and 0.3: 1. It was. As a negative control, a cellulo medium was used, and positive control cells were incubated with 1% Triton X. After 4 hours incubation at 37 ° C. in a V-bottom 96-well plate, 70 μl of supernatant was aspirated from each well and Packard Cobra Auto-Gamma 5000 Series Count System (Meriden) , Connecticut, USA). The ratio of spontaneous release was calculated from the following formula:% ⁵¹ Cr release = (sample-spontaneous) / (maximum release-spontaneous) x 100.
During the priority year, further experiments were performed with primary cells from healthy donors.

Culture of primary donor cells Luffy coat cells were obtained from a donor in a healthy blood bank at Karolinska University Hospital (Hoddinge, Stockholm, Sweden) and started to culture on the same day (day 0). PBMC were isolated by gradient centrifugation using Lymphoprep (Lymphoprep, Nyegaard, Oslo, Norway). After washing twice with phosphate buffered saline (PBS) (Gibco), cell viability was assessed by trypan blue dye exclusion and cells were cultured in 6-well culture dishes (Falcon, Becton Dickinson). (Le Pont de Clix, France)) was plated at 0.25 × 10 ⁶ cells / ml. In all cultures, 5% human serum (Biowhittaker, Cambrex BioScience, Walkersville, MD, USA), 500 IU / ml interleukin-2 (IL-2) ( Peprotech, New Jersey, USA) and Cellulo SCGM medium supplemented with 10 ng / ml anti-CD3 antibody, OKT-3 (Ortho Biotech Inc., Raritan, NJ, USA) Celgenics, Freiburg, Germany) were used. On day 5, OKT-3 was washed away, after which the cells were cultured in Serulos medium (OKT-3 free) supplemented with 500 IU / ml IL-2 and 5% human serum. Next, every 1-2 days, fresh media was added to the culture until day 21. The absolute cell number (ACC) of the subset of cells was obtained by multiplying the percentage of the subset of cells by the total number of cells in culture at the same time point.

Transduction of NK cells with retroviruses Stablely transduced retrovirus-producing cell lines producing SF91g-IL2wt, SF91g-IL2-L, SF91g-IL2ER, and SF91g-GN3 (GFP control) retroviral vectors Grow in DMEM supplemented with 10% FCS. Once the cells were somewhat confluent, fresh fresh medium was added for 24 hours. Finally, the supernatant was collected, filtered through a 0.45 μm filter (Millipore, Billerica, Massachusetts, USA) and frozen at −70 ° C. The collected and collected supernatant had a titer of 0.5 × 10 ⁶ virus particles / ml (measured with HeLa cells). Viral particles produced from this producer cell line contain the GALV (Gibbon Ape leukemia virus) envelope. The medium was centrifuged at 1.000 g for 2 hours at room temperature in the presence of 8 μg / ml polybrene (Sigma) and 500 U / ml IL-2, and the supernatant containing retrovirus (3 superinfections (MOI 3 All transductions were performed by exchanging in)). After centrifugation, the supernatant was exchanged with NK cell medium (containing Sergro medium, 500 U / ml IL-2, and 5% human serum) and the cells were allowed to grow until day 21. As a control, PBMCs from the same donor were grown and mock-infected under the same conditions as the transduced cells.

result
Effect on cell proliferation by IL-2 expression in NK-92 cells Three constructs for IL-2 expression were evaluated; one expresses wild-type IL-2 and one expresses IL-2 Targeted to the cytoplasm, one target IL-2 to the endoplasmic reticulum (Figure 1). These cytoplasmic constructs were biologically inactive (data not shown).
Following transduction, cells were cultured for 4 days in the presence of IL-2 for gene expression, then washed extensively with PBS and maintained under culture conditions with or without IL-2. . Two IL-2 transduced cell lines developed by the inventors; NK92IL2WT and NK92IL2ER were selected for this experiment. As shown in FIG. 2, untransduced NK-92 control cells died on day 6 of culture after IL-2 was removed. In contrast, NK92IL2WT and NK92IL2ER showed the same growth curves as NK-92 cells added with external IL-2 (FIG. 2).
The growth characteristics of NK92IL2WT and NK92IL2ER were stable after almost one year of continuous culture.

Endoplasmic Reticulum Localized and Limited IL-2 Expression First, the functionality of the vector for expression localized to the endoplasmic reticulum was analyzed by transient transfection into Cos-7 cells. Since Cos-7 cells showed a distinct morphology in microscopic imaging, they were selected to monitor the expression of the transduced protein. Indirect immunofluorescence of Cos-7 cells transduced with IL-2ER using a monoclonal rat anti-human IL-2 antibody showed bright green staining with localization in the endoplasmic reticulum. It was. (Data not shown).

Expression of IL-2 in the endoplasmic reticulum does not show bystander growth promotion for neighboring cells. We subsequently leaked from a cell transduced with a small amount of IL-2 into the surrounding medium. In addition, we sought to investigate whether it could be sufficient to promote the growth of parental NK-92 cells. To that end, the same number of NK-92 IL-2 modified NK-92 cell lines and GFP modified NK-92 cell lines were co-cultured and evaluated for proliferation. Forty-eight hours after culturing, the proportion of NK-92GFP cells was significantly reduced (45%) when mixed with NK92IL2ER. In contrast, enhanced proliferation was observed when mixed with NK92IL2WT cells (FIG. 3). Two days after culturing, the ratio of both groups still retains approximately 50% of the initial, indicating that NK92IL2WT secretes IL-2 into the supernatant and promotes NK92GFP survival. Indicates that

The amount of IL-2 in the supernatant was quantified by enzyme-linked immunosorbent assay. The IL-2 level detected in the supernatant from NK92IL2ER cells was 3.2 pg / ml / 10 ⁶ cells / 48 hours (FIG. 4). This is comparable to the background (including Sergro ^(R) medium, FCS). The IL-2 concentration in the supernatant from NK92IL2WT is 18.3 pg / ml / 10 ⁶ cells / 48 hours, and the supernatant from the T cells activated with anti-CD3 antibody (control) is 40.3 pg. / Ml / 10 ⁶ cells / 48 hours. Preliminary laboratory data indicate that 30 ng / ml (500 IU / ml) of IL-2 results in optimal proliferation in unmodified NK-92 cells (data not shown).

The genetically modified IL-2 modified NK-92 cell population showed a similar cytotoxic effect compared to the parental NK-92 cell line , the NK92IL2WT and NK92IL2ER lines were labeled with ⁵¹ Cr. K562 cells could be lysed at a level comparable to the parental NK-92 cells. (FIG. 5). With a 1: 1 effector: target ratio, NK-92 cells were 59% compared to an average of 59.2% (NK92IL2WT) and an average of 46.4% (NK92IL2ER) cytotoxic activity. Obtained.

  Preliminary experiments conducted on primary NK cells grown from donor PBMC during the priority year, in the absence of externally added IL-2, transfected IL2wt (SEQ ID NO: 8), and The IL2ER (SEQ ID NO: 10) gene was demonstrated to be capable of promoting the growth of primary human NK cells.

  Although the present invention has been described in terms of preferred embodiments, which constitute the best mode presently known to the inventors, it is understood that various changes and modifications that may be apparent to those skilled in the art are This may be done without departing from the scope of the invention as set forth in the claims appended hereto.

References
1. Trinchieri G Biology of natural killer cells. Adv Immunol. 1989; 47: 187-376
2. Unanue ER Studies in listeriosis show the strong symbiosis between the innate cellular system and the T-cell response. Immunol Rev. 1997; 158: 11-25
3. Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP Natural killer cells in antiviral defense: function and regulation by innate cytokines.Annu Rev Immunol. 1999; 17: 189-220
4. Orange JS, Fassett MS, Koopman LA, Boyson JE, Strominger JL Viral evasion of natural killer cells. Nat Immunol. 2002; 3: 1006-1012
5. Wu J, Lanier LL Natural killer cells and cancer.Adv Cancer Res. 2003; 90: 127-156
6. Kiessling R, Klein E, Wigzell H “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol. 1975; 5: 112-117
7. Seaman WE, Sleisenger M, Eriksson E, Koo GC Depletion of natural killer cells in mice by monoclonal antibody to NK-1.1. Reduction in host defense against malignancy without loss of cellular or humoral immunity. J Immunol. 1987; 138: 4539 -4544
8. Kim S, Iizuka K, Aguila HL, Weissman IL, Yokoyama WM In vivo natural killer cell activities revealed by natural killer cell-deficient mice.Proc Natl Acad Sci US A. 2000; 97: 2731-2736
9. Lowdell MW Natural killer cells in haematopoietic stem cell transplantation. Transfus Med. 2003; 13: 399-404
10. Parham P, McQueen KL Alloreactive killer cells: hindrance and help for haematopoietic transplants. Nat Rev Immunol. 2003; 3: 108-122
11. Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood. 1999; 94: 333-339
12. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood. 2002; 100: 1935-1947
13. Velardi A, Ruggeri L, Moretta A, Moretta L NK cells: a lesson from mismatched hematopoietic transplantation.Trends Immunol. 2002; 23: 438-444
14.Guven H, Gilljam M, Chambers BJ, et al. Expansion of natural killer (NK) and natural killer-like T (NKT) -cell populations derived from patients with B-chronic lymphocytic leukemia (B-CLL): a potential source for cellular immunotherapy. Leukemia. 2003; 17: 1973-1980
15. Lauwerys BR, Garot N, Renauld JC, Houssiau FA Cytokine production and killer activity of NK / T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18.J Immunol. 2000; 165: 1847-1853
16. Parrish-Novak J, Dillon SR, Nelson A, et al. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.Nature. 2000; 408: 57-63
17. Strengell M, Matikainen S, Siren J, et al. IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells.J Immunol. 2003; 170: 5464-5469
18. Liu CC, Perussia B, Young JD The emerging role of IL-15 in NK-cell development.Immuno Today. 2000; 21: 113-116
19. Rosenberg SA, Lotze MT, Muul LM, et al. Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer.N Engl J Med. 1985; 313: 1485-1492
20. Stein RC, Malkovska V, Morgan S, et al. The clinical effects of prolonged treatment of patients with advanced cancer with low-dose subcutaneous interleukin-2. Br J Cancer. 1991; 63: 275-278
21. Atkins MB, Lotze MT, Dutcher JP, et al.High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol. 1999; 17: 2105-2116
22. Chada S, Ramesh R, Mhashilkar AM Cytokine- and chemokine-based gene therapy for cancer. Curr Opin Mol Ther. 2003; 5: 463-474
23. Panelli MC, White R, Foster M, et al. Forecasting the cytokine storm following systemic interleukin (IL) -2 administration. J Transl Med. 2004; 2:17
24. Maas RA, Dullens HF, Den Otter W Interleukin-2 in cancer treatment: disappointing or (still) promising? A review. Cancer Immunol Immunother. 1993; 36: 141-148
25. Weisdorf DJ, Anderson PM, Blazar BR, et al. Interleukin 2 immediately after autologous bone marrow transplantation for acute lymphoblastic leukemia--a phase I study.Transplantation. 1993; 55: 61-66
26. Ochoa JB, Curti B, Peitzman AB, et al. Increased circulating nitrogen oxides after human tumor immunotherapy: correlation with toxic hemodynamic changes. J Natl Cancer Inst. 1992; 84: 864-867
27. Glauser FL, DeBlois G, Bechard D, et al. Cardiopulmonary toxicity of adoptive immunotherapy. Am J Med Sci. 1988; 296: 406-412
28. Ardizzoni A, Bonavia M, Viale M, et al. Biologic and clinical effects of continuous infusion interleukin-2 in patients with non-small cell lung cancer.Cancer. 1994; 73: 1353-1360
29. Miller JS, Tessmer-Tuck J, Blake N, et al. Endogenous IL-2 production by natural killer cells maintain cytotoxic and proliferative capacity following retroviral-mediated gene transfer.Exp Hematol. 1997; 25: 1140-1148
30. Gong JH, Maki G, Klingemann HG Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia. 1994; 8: 652-658
31. Nagashima S, Mailliard R, Kashii Y, et al. Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo. Blood; 1998; 91: 3850-3861
32. Tam YK, Maki G, Miyagawa B, et al. Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy. Hum Gene Ther. 1999; 10: 1359-1373
33. Malek TR, Bayer AL Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol. 2004; 4: 665-674
34. Laker C, Stocking C, Bergholz U, et al. Autocrine stimulation after transfer of the granulocyte / macrophage colony-stimulating factor gene and autonomous growth are distinct but interdependent steps in the oncogenic pathway.Proc Natl Acad Sci US A. 1987; 84: 8458-8462
35. Browder TM, Abrams JS, Wong PM, Nienhuis AW Mechanism of autocrine stimulation in hematopoietic cells producing interleukin-3 after retrovirus-mediated gene transfer. Mol Cell Biol. 1989; 9: 204-213

A schematic representation of a plasmid expressing a modified form of IL-2 is shown. FIG. 6 is a bar graph showing the growth of IL-2 transduced NK-92 cells and parental NK-92 cells in short term (6 days) culture. It is a bar graph which shows the result of coculturing NK-92GFP and the NK-92 modified cell which expresses IL-2 by the ratio of 1: 1. 2 is a bar graph illustrating in vitro genetically modified NK-92 IL-2 production. Shows the cytotoxicity of parental and genetically modified cells against K-562 cells in a 4 hour ⁵¹ Cr release assay.

Claims (20)

A method of immunotherapy using a genetically modified cell that expresses a substantially physiological level of IL-2, wherein the mammalian cell that expresses IL-2 comprises:
Selecting or constructing an IL-2 gene;
Producing a retroviral vector having the IL-2 gene;
Recover cells from the donor or patient,
Genetically modifying the cells,
Such a method, optionally produced by a method consisting of selecting a genetically modified cell, wherein said cell is administered to a patient in need thereof.   The method according to claim 1, wherein the IL-2 gene is a gene encoding the protein represented by SEQ ID NO: 11 or a functional equivalent thereof.   The method according to claim 1, wherein the IL-2 gene is a gene encoding the protein represented by SEQ ID NO: 13 or a functional equivalent thereof.   The method according to claim 1, wherein the IL-2 gene is a gene selected from SEQ ID NO: 8, SEQ ID NO: 10, and functional equivalents thereof.   The method according to any one of claims 1 to 4, wherein the mammalian cell is selected from natural killer (NK) cells and T cells.   6. The method of claim 5, wherein the mammalian cell is a natural killer (NK) cell.   2. The method of claim 1, wherein the IL-2 gene is modified such that IL-2 is expressed toward the endoplasmic reticulum of the cell. A method of producing a genetically modified cell that expresses a substantially physiological level of IL-2, comprising:
Selecting or constructing an IL-2 gene;
Producing a retroviral vector having the IL-2 gene;
Recover cells from the donor or patient,
Genetically modifying the cells,
Optionally, select genetically modified cells,
The above method consisting of:   The method according to claim 8, wherein the IL-2 gene is a gene encoding the protein represented by SEQ ID NO: 11 or a functional equivalent thereof.   The method according to claim 8, wherein the IL-2 gene is a gene encoding the protein represented by SEQ ID NO: 13, or a functional equivalent thereof.   The method according to claim 8, wherein the IL-2 gene is a gene selected from SEQ ID NO: 8, SEQ ID NO: 10, and functional equivalents thereof.   A transgenic mammalian cell capable of producing IL-2, obtained by the method according to any one of claims 8 to 11.   A transgenic mammalian cell obtained by the method according to any one of claims 8-11, wherein the cells in an unmodified state are IL-2 dependent with respect to proliferation and are expressed in significant amounts. The cell, wherein the transgenic cell produces IL-2 in an amount sufficient to maintain proliferation without the need for external IL-2.   14. The transgenic mammalian cell of claim 13, wherein IL-2 is expressed and retained in the endoplasmic reticulum of the cell.   15. A method of treating cancer comprising administering to a patient a therapeutically effective and physiologically acceptable amount of the transgenic cells of any one of claims 12-14.   16. A method according to any one of claims 1 to 7 or 15, wherein the cells are harvested from the patient, transfected and returned to the same patient.   16. The method of any one of claims 1-7 or 15, wherein the cells are harvested from a donor, transfected and administered to a patient.   The use of transgenic cells is one of additional or supplemental therapies and is performed before, after, or substantially simultaneously with other therapies. The method according to any one of the above.   16. The method according to any one of claims 1 to 7 or 15, wherein the use of transgenic cells is one of the primary therapies in the treatment of cancer.   The method according to any one of claims 1 to 7, wherein the immune promotion is one of treatments in the treatment or prevention of infection.

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